Echinacea species represent one of the most widely used immunostimulatory botanicals in the US herbal market. However, clinical evaluation of efficacy has met with inconsistent results that are difficult to interpret because they have used a wide variety of chemically ill-defined preparations. In addition, despite decades of research, it i not known if Echinacea's main therapeutic action on colds and flu is immunostimulatory, anti-inflammatory or a combination of both. Animal research indicates that this botanical exerts both therapeutic actions and Echinacea alkylamides represent the major anti-inflammatory component. Echinacea polysaccharides, once thought to be responsible for the immune enhancing action of this plant, are present at such low levels and exhibit extremely weak in vitro innate immune activation potential and thus are not likely to be therapeutically relevant. We have found that the bacterial components lipopolysaccharides and Braun-type lipoproteins are major in vitro and in vivo innate immune cell activators within Echinacea and other immune enhancing botanicals and those endophytic bacteria represent the principal source of these components. Differences in the levels of these two bacterial components are responsible for the substantial variation observed (~100-fold) in innate immune cell activation from E. purpurea extracts of material obtained from six major growers/commercial suppliers in North America. This substantial variability in bacterial component content/activity could be a factor contributing to the inconsistent outcomes exhibited in Echinacea clinical trials since immune related benefits have been demonstrated in studies employing orally administered bacterial preparations such as probiotics. Our hypothesis is that endophytic bacteria within E. purpurea are a major source of LPS and Brawn type lipoproteins and variation in the amount/type of these endophytes are responsible for differences in the immune enhancing potential of diverse Echinacea plant material. Our preliminary studies have identified 23 bacterial endophytic species within E. purpurea and extracts from these isolates varied substantially (up to 10,000-fold) in their ability to activate macrophages in vitro. Aim 1 will identify which bacterial endophytes within E. purpurea are principal contributors to the activity exhibited by the plant material. Aim 2 will determine if the in vitro activity and in vivo action of E. purpurea on several immune parameters in mice can be mimicked by the oral ingestion of endophytic bacteria equivalent in amount/type to that found in this plant material. Aim 3 will identify agronomic factors influencing E. purpurea plant activity and will define shifts in endophyte content/activity responsible for activity change. Accomplishing these objectives will provide a greater understanding of how Echinacea influences the innate immune system and will impact CAM research by providing the information and tools to more appropriately standardize Echinacea products for use in clinical studies. This research will also add to the growing body of evidence that indicates that the oral consumption of certain types of bacteria or bacterial components can have a major impact on human health.